Unit 2: Preparation of plant tissue culture media - Subjective Questions

Plant tissue culture media is a chemically defined nutrient medium that provides all the essential inorganic and organic nutrients required for the growth, differentiation, and multiplication of plant cells, tissues, and organs under aseptic (in vitro) conditions.

Significance:

• Supplies macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Mn, Zn, B, Cu, Mo, Co) essential for metabolism.
• Provides a carbon source (usually sucrose) as energy since cultured tissues are often non-photosynthetic.
• Contains vitamins (thiamine, pyridoxine, nicotinic acid) and amino acids for cellular functions.
• Supplies plant growth regulators (auxins, cytokinins) to control differentiation.
• Enables controlled morphogenesis, callus induction, organogenesis, and somatic embryogenesis.
• Ensures reproducibility and standardization of culture experiments.

The medium composition can be modified to achieve specific goals such as shoot induction, root induction, or callus proliferation.

A complete plant tissue culture medium consists of the following major components:

1. Inorganic Nutrients:

• Macronutrients (required in > 0.5 mmol/L): Nitrogen (, ), Phosphorus, Potassium, Calcium, Magnesium, Sulphur.
• Micronutrients (required in trace amounts): Iron (as Fe-EDTA), Manganese, Zinc, Boron, Copper, Molybdenum, Cobalt.

2. Carbon and Energy Source:

• Sucrose (2–5%) is most commonly used; glucose and fructose can also be used.

3. Organic Supplements:

• Vitamins: Thiamine (B1), Pyridoxine (B6), Nicotinic acid.
• Amino acids: Glycine, glutamine, asparagine.
• Myo-inositol: promotes cell division.

4. Plant Growth Regulators (PGRs):

• Auxins: IAA, IBA, NAA, 2,4-D.
• Cytokinins: BAP, Kinetin, Zeatin.
• Gibberellins and Abscisic acid in specific cases.

5. Solidifying Agent:

• Agar (0.6–1.0%) or Gelrite/Phytagel for semi-solid media.

6. Undefined Organic Complexes (optional):

• Coconut water, casein hydrolysate, yeast extract, banana pulp.

The pH is adjusted to 5.6–5.8 before autoclaving.

Conclusion: Solid media are preferred for morphogenesis and maintenance, whereas liquid media are ideal for rapid cell multiplication and suspension cultures.

MS medium, developed by Murashige and Skoog (1962), is the most widely used plant tissue culture medium due to its high concentration of nitrate, potassium, and ammonium salts.

Composition Highlights:

• Macronutrients: High levels of (1650 mg/L) and (1900 mg/L), plus , , .
• Micronutrients: , , , , , , .
• Iron source: + (chelated iron).
• Organic components: Myo-inositol (100 mg/L), thiamine, pyridoxine, nicotinic acid, glycine.
• Carbon source: Sucrose (30 g/L).
• pH: 5.6–5.8.

Importance:

• Supports growth of a wide range of plant species.
• Its high salt content meets nutritional demands of rapidly dividing cells.
• Serves as the basal medium that can be supplemented with various PGRs.
• Provides reproducibility and standardization in experiments worldwide.

Preparation of Semi-Solid MS Medium:

Step 1 – Preparation of stock solutions:

• Prepare separate stock solutions of macronutrients, micronutrients, iron-EDTA, vitamins, and PGRs to avoid precipitation and improve accuracy.

Step 2 – Measuring water:

• Take about 70–80% of the required final volume of distilled/double-distilled water in a beaker.

Step 3 – Adding stocks:

• Add measured aliquots of each stock solution in order: macronutrients → micronutrients → iron → vitamins.

Step 4 – Adding carbon source:

• Add sucrose (30 g/L) and stir until dissolved.

Step 5 – Adding PGRs:

• Add required auxins and cytokinins from prepared stocks.

Step 6 – Volume adjustment:

• Make up the volume to the final level with distilled water.

Step 7 – pH adjustment:

• Adjust pH to 5.6–5.8 using 0.1 N NaOH or 0.1 N HCl.

Step 8 – Adding agar:

• Add agar (0.7–0.8%) and heat to dissolve (for solid media).

Step 9 – Dispensing:

• Pour into culture vessels/test tubes and cap them.

Step 10 – Sterilization:

• Autoclave at 121°C, 15 psi for 15–20 minutes.

Step 11 – Cooling and storage:

• Allow media to solidify and store in a clean, cool environment until use.

Stock solutions are concentrated solutions of individual media components (or groups of related components) prepared in advance and diluted appropriately during final media preparation.

Why stock solutions are prepared:

• Convenience: Avoids repeated weighing of small quantities each time.
• Accuracy: Allows precise pipetting instead of weighing tiny amounts of chemicals.
• Time-saving: Speeds up routine media preparation.

Why they are stored separately:

• Prevents precipitation: Certain salts (e.g., calcium and phosphate) form insoluble precipitates when mixed in concentrated form.
• Chemical stability: Iron is prepared separately as Fe-EDTA chelate to prevent oxidation and precipitation.
• Stability of organics: Vitamins and growth regulators are heat/light sensitive and stored refrigerated or frozen.

Typical stock categories:

• Macronutrient stock (usually 10× or 20×)
• Micronutrient stock (100× or 1000×)
• Iron-EDTA stock
• Vitamin stock
• Individual PGR stocks

Stocks are stored at 4°C (short term) or –20°C (long term) and labelled clearly with concentration and date.

Plant Growth Regulators (PGRs) are added in small quantities to media to control growth and differentiation. The two most important classes are auxins and cytokinins.

1. Auxins:

• Examples: IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), NAA (Naphthalene acetic acid), 2,4-D (2,4-Dichlorophenoxyacetic acid).
• Functions:
  • Promote cell division and elongation.
  • Induce root formation (rhizogenesis).
  • Stimulate callus induction (especially 2,4-D).
  • Regulate apical dominance.

2. Cytokinins:

• Examples: BAP (6-Benzylaminopurine), Kinetin, Zeatin, 2-iP.
• Functions:
  • Promote cell division.
  • Induce shoot proliferation and multiplication.
  • Break apical dominance and promote axillary bud growth.
  • Delay senescence.

Auxin : Cytokinin Ratio (Skoog & Miller concept):

• High auxin : low cytokinin → root formation
• Low auxin : high cytokinin → shoot formation
• Balanced ratio → callus proliferation

Thus, by manipulating the ratio, the direction of morphogenesis can be controlled.

Sterilization is essential to maintain aseptic conditions and prevent microbial contamination. Various methods are used depending on the material:

1. Physical Methods:

• Moist heat (Autoclaving): Media, water, glassware sterilized at 121°C, 15 psi for 15–20 min — most common method.
• Dry heat (Hot air oven): Glassware and metal instruments at 160–180°C for 2–3 hours.
• Flame sterilization: Instruments (forceps, scalpels) dipped in ethanol and flamed.
• Incineration: Destruction of contaminated material.
• UV radiation: Sterilization of laminar air flow chamber surfaces and air.

2. Chemical Methods:

• Surface sterilization of explants: Using sodium hypochlorite (0.5–1%), mercuric chloride (, 0.1%), ethanol (70%).
• Chemical wipes: Ethanol/formaldehyde for work surfaces.

3. Filtration (Membrane filtration):

• Used for heat-labile components (certain vitamins, growth regulators, antibiotics) using 0.22 µm membrane filters.

4. Aseptic technique:

• Working inside a laminar air flow chamber with HEPA-filtered air.

Proper combination of these methods ensures contamination-free cultures.

Principle of Autoclaving:

• Autoclaving is a method of sterilization by moist heat under pressure.
• Water boils at higher temperature under increased pressure. At 15 psi (pounds per square inch), water boils at 121°C.
• The saturated steam at this temperature causes denaturation and coagulation of microbial proteins, killing bacteria, fungi, and spores.

Standard conditions:

• Temperature: 121°C
• Pressure: 15 psi (≈ 1.05 kg/cm²)
• Time: 15–20 minutes

Procedure:

1. Fill the autoclave chamber with sufficient water.
2. Place the media containers (loosely capped to allow steam entry) inside.
3. Close and seal the lid tightly.
4. Heat until steam is generated; allow air to escape completely (air removal is critical).
5. Close the exhaust valve and let pressure build to 15 psi.
6. Maintain 121°C for 15–20 minutes.
7. Switch off and allow pressure to fall to zero naturally.
8. Open the lid carefully and remove the sterilized media.

Precautions:

• Do not overload the chamber.
• Ensure complete air removal.
• Do not open until pressure reaches zero.
• Heat-labile substances must be filter-sterilized separately.

Heat-labile compounds are substances that get degraded or denatured by the high temperature (121°C) of autoclaving, losing their biological activity.

Examples of heat-labile compounds:

• Certain plant growth regulators (GA3, zeatin, IAA, ABA).
• Some vitamins (thiamine, pantothenic acid).
• Antibiotics (cefotaxime, kanamycin).
• Enzymes and amino acids like glutamine.

Why they cannot be autoclaved:

• High temperature breaks chemical bonds and alters molecular structure.
• This causes loss of function — e.g., growth regulators lose activity, antibiotics become ineffective.

Method used – Membrane (Filter) Sterilization:

1. The compound is dissolved in sterile water/buffer.
2. The solution is passed through a membrane filter of pore size 0.22 µm (or 0.45 µm).
3. Microorganisms are physically retained on the filter while sterile solution passes through.
4. The filtered solution is added aseptically to the previously autoclaved, cooled (≈ 40–45°C) medium under a laminar air flow.

Advantages:

• No heat damage to sensitive molecules.
• Retains full biological activity.

This ensures both sterility and preservation of the compound's function.

pH is a critical parameter in media preparation that affects nutrient availability, gelling, and growth.

Optimal pH: The pH of most plant tissue culture media is adjusted to 5.6–5.8 before autoclaving.

Importance of correct pH:

• Nutrient solubility and availability: At improper pH, salts (especially iron and phosphates) may precipitate, making them unavailable.
• Agar gelling: Agar does not solidify properly if the pH is too low (< 4.5); it remains soft and watery.
• Uptake of nutrients and PGRs: pH influences membrane transport and ion uptake by cultured tissues.
• Enzyme activity: Cellular metabolic enzymes function optimally within a narrow pH range.

Effects of incorrect pH:

• Too acidic: Poor gelling, reduced growth.
• Too alkaline: Precipitation of salts, hardened medium, inhibited growth.

Adjustment procedure:

• pH is measured with a calibrated pH meter and adjusted using 0.1 N NaOH (to raise) or 0.1 N HCl (to lower) before adding agar and autoclaving.

Note: pH may drop slightly (0.3–0.5 units) after autoclaving, which is accounted for during preparation.

Agar is the most commonly used solidifying/gelling agent in plant tissue culture media.

Nature and Role of Agar:

• Agar is a polysaccharide obtained from red algae (seaweeds like Gelidium and Gracilaria).
• It melts at around 100°C and solidifies at 40–45°C, forming a firm gel.
• Used at a concentration of 0.6–1.0% (6–10 g/L).
• Provides a solid support on which the explant is placed, keeping it in contact with nutrients while allowing partial aeration.
• It is biologically inert and does not react with media components.

Advantages of Agar:

• Non-toxic, stable, and does not digest by plant enzymes.
• Clear enough to observe root/microbial growth.

Limitations:

• Expensive, may contain impurities.
• Gel strength affected by pH.

Alternatives to Agar:

• Gelrite / Phytagel (gellan gum) – produces clearer, firmer gel at lower concentration (0.2–0.4%).
• Agarose – purified form for sensitive cultures.
• Starch, alginate, and Guar gum – used in some special applications.

The choice depends on cost, clarity requirement, and sensitivity of cultures.

Conclusion: MS medium is a high-salt, general-purpose medium suited for a wide range of applications, whereas White's medium is a low-salt medium primarily used for root cultures and tissues sensitive to high salt concentrations.

Liquid Media Preparation:

• The procedure is identical to solid media preparation except that agar (or any gelling agent) is NOT added.

Steps:

1. Prepare and mix all stock solutions (macro, micro, iron, vitamins) in distilled water.
2. Add sucrose (30 g/L) and required PGRs.
3. Make up the final volume.
4. Adjust pH to 5.6–5.8.
5. Dispense into culture flasks/conical vessels.
6. Autoclave at 121°C, 15 psi for 15–20 minutes.

Establishment of Suspension Culture:

• A friable callus is transferred into the sterilized liquid medium.
• The flask is placed on an orbital/rotary shaker (80–150 rpm) to:
  • Ensure uniform distribution of cells.
  • Provide adequate aeration/oxygen supply.
  • Break cell aggregates into a fine suspension.
• Incubation at 25 ± 2°C with controlled light.

Applications of Liquid/Suspension Cultures:

• Rapid biomass production.
• Production of secondary metabolites.
• Study of cell growth kinetics.
• Protoplast and single-cell studies.

Precaution: Since explants are submerged, constant agitation is essential to prevent hypoxia and hyperhydricity.

Organic supplements are organic compounds added to media to enhance growth and differentiation of cultured tissues, beyond the basic inorganic nutrients.

Categories and Functions:

1. Vitamins:

• Thiamine (B1): Essential; involved in carbohydrate metabolism.
• Pyridoxine (B6): Coenzyme in amino acid metabolism.
• Nicotinic acid (B3): Involved in redox reactions.

2. Amino Acids:

• Glycine, Glutamine, Asparagine, Arginine: Provide organic nitrogen and promote growth of cells and embryos.

3. Myo-inositol:

• A sugar alcohol that promotes cell division and growth; commonly added at 100 mg/L.

4. Undefined (Complex) Organic Supplements:

• Coconut water: Rich in cytokinins, amino acids — promotes cell division.
• Casein hydrolysate: Source of amino acids.
• Yeast extract & Malt extract: Provide vitamins and nitrogen.
• Banana pulp, potato extract: Used in orchid cultures.

Importance:

• Enhance growth rates and morphogenetic response.
• Supply growth-limiting organic factors.
• Improve success of difficult-to-culture species.

Note: Undefined supplements cause batch-to-batch variation, so defined media are preferred for reproducibility.

Aseptic technique refers to the set of practices used to prevent contamination of cultures by microorganisms (bacteria, fungi, yeasts) during handling of media and explants.

Importance:

• Cultured plant tissues have no defense against microbes; contamination causes rapid culture loss.
• Microorganisms compete for nutrients and release toxins, killing tissues.
• Essential for reproducibility and reliable results.

Laminar Air Flow (LAF) Chamber:

• A workstation providing a sterile working environment using filtered air.

Working Principle:

• Air is drawn in and passed through a HEPA (High Efficiency Particulate Air) filter that removes ~99.97% of particles ≥ 0.3 µm.
• Filtered sterile air flows in a laminar (unidirectional) stream across the work surface, preventing entry of airborne contaminants.

Features & Use:

• Equipped with a UV lamp for surface sterilization before use (switched off during work).
• Work surface wiped with 70% ethanol.
• Instruments flame-sterilized inside the cabinet.
• Operator works with clean hands, sleeves, and avoids obstructing air flow.

Types: Horizontal and Vertical LAF chambers.

Proper use of the LAF chamber combined with aseptic technique ensures contamination-free tissue culture.

Surface sterilization is the process of removing surface microorganisms from plant explants (leaf, stem, node, seed, etc.) without damaging plant tissue, before inoculation onto media.

General Procedure:

1. Washing: Explant washed thoroughly under running tap water to remove dust and debris.
2. Detergent treatment: Dipped in dilute detergent (e.g., Tween-20) to remove surface dirt and remove surface tension.
3. Ethanol treatment: Dipped in 70% ethanol for 30–60 seconds (kills surface microbes rapidly).
4. Main sterilant treatment: Treated with a surface sterilant such as:
   • Sodium hypochlorite (NaOCl, 0.5–1%) for 10–15 min, OR
   • Mercuric chloride (, 0.1%) for 2–10 min (highly toxic; use carefully), OR
   • Hydrogen peroxide / Calcium hypochlorite.
5. Rinsing: Rinsed 3–4 times with sterile distilled water to remove traces of sterilant.
6. Inoculation: Sterilized explant trimmed and placed on medium under LAF.

Key Points:

• Concentration and duration must balance effective sterilization vs. tissue damage.
• All steps after ethanol treatment are done under aseptic conditions.

Proper surface sterilization is crucial for establishing contamination-free cultures.

Explanation:

• Defined media allow precise control and reproducible results, essential for research.
• Undefined media contain natural extracts rich in unidentified growth factors that improve growth of difficult species but introduce variability.

Conclusion: Defined media are chosen for reproducible, controlled studies, while undefined supplements are added when their growth-promoting benefits outweigh the variability.

Several precautions must be observed to ensure successful, contamination-free media:

During Media Preparation:

• Use double-distilled or deionized water of high purity.
• Use analytical grade chemicals accurately weighed.
• Add stock solutions in the correct order to avoid precipitation.
• Dissolve sucrose and salts completely before pH adjustment.
• Adjust pH to 5.6–5.8 before adding agar and before autoclaving.
• Prepare and store heat-labile PGRs and vitamins separately (filter-sterilize).

During Sterilization (Autoclaving):

• Loosely cap containers to allow steam penetration.
• Ensure complete air removal from the autoclave.
• Do not overload the chamber.
• Maintain 121°C, 15 psi for 15–20 min.
• Avoid over-autoclaving which degrades sucrose and vitamins.
• Allow pressure to fall to zero naturally before opening.

General Precautions:

• Add filter-sterilized components only after media cools to ~40–45°C.
• Store media in a clean, dust-free area.
• Check media for contamination before use (incubate 2–3 days).
• Maintain aseptic conditions using LAF and sterile glassware.

Strict adherence prevents nutrient degradation and microbial contamination.

Culture media in plant tissue culture can be classified based on physical consistency into three main types:

1. Solid (Semi-Solid) Media:

• Prepared by adding a gelling agent (agar 0.6–1.0% or Gelrite).
• Explant is placed on the surface.
• Applications: Callus culture, shoot/root organogenesis, micropropagation, germplasm storage, and routine subculturing.

2. Liquid Media:

• No gelling agent added.
• Requires agitation (shaking) for aeration, or a support system (filter paper bridge, rafts).
• Applications: Cell suspension culture, biomass/secondary metabolite production, protoplast culture, single-cell studies.

3. Biphasic (Two-Phase) Media:

• Combination of a solid layer overlaid with a liquid layer in the same vessel.
• Nutrients diffuse from solid to liquid phase, providing fresh nutrients to cultures.
• Applications: Enhancing growth and productivity, replenishing depleted nutrients.

Additional classification based on purpose:

• Induction media (callus induction)
• Multiplication media (shoot proliferation)
• Rooting media (root induction)

Conclusion: The choice of media consistency depends on the culture type and the desired morphogenetic outcome, with solid media used for morphogenesis and liquid media for rapid cell multiplication.